Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus |
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Authors: | Bollati Michela Milani Mario Mastrangelo Eloise Ricagno Stefano Tedeschi Gabriella Nonnis Simona Decroly Etienne Selisko Barbara de Lamballerie Xavier Coutard Bruno Canard Bruno Bolognesi Martino |
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Institution: | 1 Department of Biomolecular Sciences and Biotechnology, CNR-INFM and CIMAINA, University of Milano, Via Celoria 26, 20133 Milan, Italy 2 DIPAV, Biochemistry Section, University of Milano, via Celoria 10, 20133 Milan, Italy 3 Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098, AFMB-CNRS-ESIL, Case 925, 163 Avenue de Luminy, 13288 Marseille, France 4 Unité des Virus Emergents, Faculté de Médecine, 27 Bd Jean Moulin, 13005 Marseille, France |
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Abstract: | The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′-5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7MeGpppG, and N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities. |
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Keywords: | NS nonstructural proteins RTPase RNA triphosphatase GMP guanosine monophosphate GTase guanylyltransferase N7-MTase guanine N7-methyltransferase AdoMet l-methionine" target="_blank">S-adenosyl-l-methionine MVEvMTase Murray Valley encephalitis virus MTase DvMTase dengue virus MTase HBS high-affinity binding site WvMTase Wesselsbron virus MTase AdoHcy l-homocysteine" target="_blank">S-adenosyl-l-homocysteine LBS low-affinity binding site RdRp RNA-dependent RNA polymerase PDB Protein Data Bank GTP guanosine triphosphate |
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